Our long-term objectives are to determine the cellular and molecular bases for skeletal development and repair. Growing evidence indicates that the program of embryonic development is recapitulated in response to injury. The clinical significance of this observation is clear: by understanding the molecular and cellular basis of skeletogenesis, we can develop therapeutic strategies for the treatment of musculoskeletal injuries, defects, and diseases in humans. To pursue these objectives, we have developed multiple methods to study bone development in the embryo including in vivo genetic approaches, and in vivo and in vitro manipulations of developing bones. Molecular insights gained from developmental studies can be directly applied to our studies of fracture repair using the various bone healing models we have developed. Through these diverse approaches we have developed a framework for studying complex cellular and molecular mechanisms controlling skeletal development and repair. The molecular mechanisms that govern the differentiation of stem cells during fracture repair, and thus govern the orderly process of skeletal tissue regeneration, remain largely unknown. Our research proposal focuses on the origins of these stem cells, and their ability to contribute to bony regeneration. Whereas the majority of the skeleton is derived from mesoderm, all of the cartilages and bones in the facial and jaw skeleton are derived from the cranial neural crest. The fact that these skeletal tissues originate from distinct lineages raises the possibility that each lineage provides a unique stem cell population that contributes to the regenerating skeletal tissue. We hypothesize that cranial skeletal injuries heal predominantly through the recruitment of neural crest-derived stem cells, whereas long bone injuries heal through the preferential recruitment of mesoderm-derived stem cells. To test these hypotheses, we will determine the contribution of marrow-derived stem cells in a mandibular fracture callus compared to the contribution of marrow-derived stem cells in a long bone fracture callus. We will determine if stem cells derived from the cranial neural crest exist in adult animals, and the extent to which these cells contribute to regeneration of neural crest-derived skeletal tissues. This project is significant in using a novel approach to investigate regulation of fracture repair and will provide valuable insights on healing defect.